The present invention is directed to methods of producing an attenuated form of bovine viral diarrhea (BVD) virus by inactivating a specific gene in the viral genome. The attenuated virus, or the mutated viral genome, can be used to produce antibody against BVD virus or in vaccines designed to protect cattle from viral infection.
Bovine viral diarrhea (BVD) virus is classified in the pestivirus genus and Flaviviridae family. It is closely related to viruses causing border disease in sheep and classical swine fever. Infected cattle exhibit xe2x80x9cmucosal diseasexe2x80x9d which is characterized by elevated temperature, diarrhea, coughing and ulcerations of the alimentary mucosa (Olafson, et al., Cornell Vet. 36:205-213 (1946); Ramsey, et al., North Am. Vet. 34:629-633 (1953)). The BVD virus is capable of crossing the placenta of pregnant cattle and may result in the birth of persistently infected (PI) calves (Malmquist, J. Am. Vet. Med. Assoc. 152:763-768 (1968); Ross, et al., J. Am. Vet. Med. Assoc. 188:618-619 (1986)). These calves are immunotolerant to the virus and persistently viremic for the rest of their lives. They provide a source for outbreaks of mucosal disease (Liess, et al., Dtsch. Tieraerztl. Wschr. 81:481-487 (1974)) and are highly predisposed to infection with microorganisms causing diseases such as pneumonia or enteric disease (Barber, et al., Vet. Rec. 117:459-464 (1985)).
BVD viruses are classified as having one of two different biotypes. Those of the xe2x80x9ccpxe2x80x9d biotype induce a cytopathic effect in cultured cells, whereas viruses of the xe2x80x9cncpxe2x80x9d biotype do not (Gillespie, et al., Cornell Vet. 50:73-79 (1960)). In addition, two major genotypes (type I and II) are recognized, both of which have been shown to cause a variety of clinical syndromes (Pellerin, et al., Virology 203:260-268 (1994); Ridpath, et al., Virology 205:66-74 (1994)).
The genome of the BVD virus is approximately 12.5 kb in length and contains a single open reading frame located between the 5xe2x80x2 and 3xe2x80x2 non-translated regions (NTRs) (Collett, et al., Virology 165:191-199 (1988)). A polyprotein of approximately 438 kD is translated from this open reading frame and is processed into viral structural and nonstructural proteins by cellular and viral proteases (Tautz, et al., J. Virol. 71:5415-5422 (1997); Xu, et al., J. Virol. 71:5312-5322 (1997); Elbers, et al., J. Virol. 70:4131-4135 (1996); and Wiskerchen, et al., Virology 184:341-350 (1991)). Among the viral enzymes that participate in this processing are the proteases Npro and NS3. Npro is the first protein encoded by the viral open reading frame and cleaves itself from the rest of the synthesized polyprotein (Stark, et al., J. Virol. 67:7088-7093 (1993); Wiskerchen, et al., Virol. 65:4508-4514 (1991)).
Among the BVD vaccines that are currently available are those in which virus has been chemically inactivated (McClurkin, et al., Arch. Virol. 58:119 (1978); Fernelius, et al., Am. J. Vet. Res. 33:1421-1431 (1972); and Kolar, et al., Am. J. Vet. Res. 33:1415-1420 (1972)). These vaccines have typically required the administration of multiple doses to achieve primary immunization, provide immunity of short duration and do not protect against fetal transmission (Bolin, Vet. Clin. North Am. Food Anim. Pract. 11:615-625 (1995)). In sheep, a subunit vaccine based upon a purified E2 protein has been reported (Bruschke, et al., Vaccine 15:1940-1945 (1997)). Unfortunately, only one such vaccine appears to protect fetuses from infection and this protection is limited to one strain of homologous virus. There is no correlation between antibody titers and protection from viral infection.
In addition, modified live virus (MLV) vaccines have been produced using BVD virus that has been attenuated by repeated passage in bovine or porcine cells (Coggins, et al., Cornell Vet. 51:539 (1961); and Phillips, et al., Am. J. Vet Res. 36:135 (1975)) or by chemically induced mutations that confer a temperature-sensitive phenotype on the virus (Lobmann, et al., Am. J. Vet. Res. 45:2498 (1984); and Lobmann, et al., Am. J. Vet. Res. 47:557-561 (1986)). A single dose of MLV vaccine has proven sufficient for immunization and the duration of immunity can extend for years in vaccinated cattle (Coria, et al., Can. J. Con. Med. 42:239 (1978)). In addition, cross-protection has been reported from calves vaccinated with MLV-type vaccines (Martin, et al., In Proceedings of the Conference Res. Workers"" Anim. Dis., 75:183 (1994)). However, safety considerations, such as possible fetal transmission of the virus, have been a major concern with respect to the use of these vaccines (Bolin, Vet. Clin. North Am. Food Anim. Pract. 11:615-625 (1995)).
A clear need exists for new and effective vaccines to control the spread of the BVD virus. Given that the disease caused by this virus is one of the most widespread and economically important diseases of cattle, such vaccines would represent a substantial advance in livestock farming.
The present invention is based upon the discovery that attenuated forms of BVD virus can be produced by deleting or inactivating the Npro protease gene. These viruses are much less infectious than their wild-type counterparts in bovine cell lines and are suitable for use in vaccines for cattle. A complete genomic sequence of one such attenuated virus is disclosed herein, and a plasmid encoding this virus, i.e., pBVDdN1, has been deposited with the American Type Culture Collection (ATCC) as ATCC No. 203354.
A. Compositions and Methods Based Upon the BVDdN1 Attenuated Virus
In its first aspect, the present invention is based upon the development of a specific attenuated BVD viral strain. The strain is produced by mutating a wild type viral genome to delete the Npro protease gene and its full-length sequence is shown in SEQ ID NO:1 and FIG. 2, from nt 39 to nt 12116. Thus, the invention is directed to a virus having a genomic sequence comprising that shown, and preferably consisting essentially of that shown. Ordinarily, the BVD virus has a genome in the form of RNA. When cloned, this will more typically be in the form of DNA. Unless otherwise indicated, the term xe2x80x9cnucleic acidxe2x80x9d refers to both BVD viral DNA and RNA sequences. For convenience, sequence listing entries only show DNA sequences but the corresponding RNA sequence for each will be readily apparent to those of skill in the art. The term xe2x80x9cconsisting essentially ofxe2x80x9d refers to sequences that are substantially the same as those specified both in terms of structure and function. Thus, the invention includes not only the sequences expressly depicted, but also corresponding sequences made by introducing insubstantial additions or substitutions. In particular, the invention includes degenerate nucleic acid sequences that encode the same BVD proteins as SEQ ID NO:1. This particular sequence, i.e., SEQ ID NO:1 from nt 39 to nt 12116, and the corresponding virus it encodes have, for convenience, been designated as the xe2x80x9cBVDdN1xe2x80x9d genome and virus. Virus can be present either as part of a larger preparation or in substantially purified form, i.e., in a form essentially free from any other viral types.
The invention includes host cells carrying a BVDdN1 nucleic acid molecule of the present invention. The term xe2x80x9chost cellsxe2x80x9d is meant to include any prokaryotic cells carrying a BVDdN1 nucleic acid molecule, and any eukaryotic cells infected with the virus or otherwise carrying a BVDdN1 nucleic acid molecule. For prokaryotic cells, the STBL2 strain of E. coli (GibcoBRL) has been found to give the best results for propagating the plasmid, and is generally preferred. For eukaryotic cells, mammalian cells such as MDBK cells (ATCC CCL-22) and RD cells (stable transformed bovine testicular cells) are generally preferred. However, other cultured cells can be used as well. The invention further includes progeny virus produced in such host cells.
The BVDdN1 virus can be used to induce the production of antibody by infecting an animal at an effective dosage, i.e., at a dosage high enough to provoke antibody production. The antibodies can be made in any of the animals normally used for this purpose (such as mice, rabbits, goats, or sheep) but, preferably, antibodies will be made in cattle. The term xe2x80x9cantibody to BVD virusxe2x80x9d as used herein refers to antibodies that react preferentially in the sense of having at least a 100-fold greater affinity for a strain of BVD virus than for any other, non-BVD virus. Although not preferred, virus can be further inactivated prior to administration to an animal using chemical treatments involving agents such as formalin, paraformaldehyde, phenol, lactopropionate, psoralens, platinum complexes, ozone or other viricidal agents. Antibodies made by these procedures are themselves included within the scope of the invention and can be isolated using techniques that are well known in the art (see e.g., Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1988)). The antibodies can be used, inter alia, in methods designed to detect the presence of BVD in biological or laboratory samples.
In another aspect, the invention is directed to a vaccine comprising the BVDdN1 virus and a veterinarily acceptable carrier. This vaccine can include any of the adjuvants and other agents typically used in such preparations. An immune response can be induced in cattle by administering the vaccine at a dosage sufficient to induce protective immunity against subsequent challenge with BVD virus. Typically, the vaccine will be administered parenterally, but other routes of administration are compatible with the invention as well. If necessary, two or more inoculations can be given at regular intervals of, for example, two to eight weeks. Standard procedures well known in the art can be used to optimize immunization protocols.
B. Compositions and Methods Based Upon BVDdN1 Genomic Nucleic Acid
Recent work has demonstrated that it is possible to prepare effective vaccines by injecting animals with nucleic acids encoding immunogens. Methods for making and administering these xe2x80x9cDNA vaccinesxe2x80x9d have been described in detail (see e.g., U.S. Pat. Nos. 5,589,466; 5,580,859; and 5,703,055) and can be applied to BVDdN1 genomic nucleic acid. Thus, in another aspect, the present invention is directed to a nucleic acid molecule, preferably in substantially purified form, comprising the sequence of SEQ ID NO:1, from nt 39 to nt 12116, or a degenerate variant thereof. In a preferred embodiment, the present invention is directed to a nucleic acid molecule, preferably in substantially purified form, consisting essentially of the sequence of SEQ ID NO:1, from nt 39 to nt 12116. As used herein, xe2x80x9csubstantially purifiedxe2x80x9d refers to a desired product that is essentially free from contaminating materials. For example, a xe2x80x9csubstantially purifiedxe2x80x9d nucleic acid molecule would be essentially free from other contaminating nucleic acid molecules and typically comprise at least 85 wt % of the nucleic acid molecules in a sample, with greater percentages being preferred. One method for determining the purity of a nucleic acid is by electrophoresing a preparation in a matrix such as polyacrylamide or agarose. Purity is evidenced by the appearance of a single band after staining. Other methods for assessing purity include chromatography and analytical centrifugation.
The BVDdN1 genomic nucleic acid can be incorporated into a vector as a distinct coding element. The phrase xe2x80x9cdistinct coding elementxe2x80x9d refers to the portion of the vector that is translated into viral polypeptide and, eventually, virus. It is distinct in the sense that it does not include any other translated elements that would substantially alter the BVDdN1 product. This vector, or the BVDdN1 nucleic acid itself, can be used to transfect a host cell in order to produce progeny attenuated virus.
The invention also includes methods of inducing the production of antibody to BVD virus by injecting the BVDdN1 nucleic acid, or a vector containing this nucleic acid, directly into an animal. Any animal capable of making antibody can be used, but cattle are generally preferred. Antibody made in this way is part of the invention and can be purified from animals and used, for example, in assays designed to detect the presence of BVD virus in culture medium or biological fluid.
Vaccines for administration to cattle can be prepared based upon the BVDdN1 genomic nucleic acid (see references cited supra), in combination with a veterinarily acceptable carrier, and used in immunization protocols optimized for inducing protective immunity against subsequent viral infection.
C. Methods of Mutating Wild Type BVD Genomes
In a more general sense, the present invention is directed to a method of modifying a genome from a substantially purified wild type BVD virus in such a manner as to make it suitable for use in a vaccine. The term xe2x80x9csubstantially purifiedxe2x80x9d as used in this context refers to a viral preparation consisting, preferably, of a single strain of BVD virus with no other types of virus being present. The main distinguishing feature of the procedure is that the genomic nucleic acid is mutated to inactivate the Npro protease gene. In this context, a gene is considered to be inactivated either if no product is made (for example, the gene is deleted), or a product is made that can no longer carry out its normal biological function (e.g., proteolytic cleavage), or a product is made that carries out its normal biological function but at a significantly reduced rate. Any method that results in the inactivation of the Npro protease can be used. For example, genomic RNA can be isolated from the wild type BVD virus, reverse transcribed to form cDNA and then cloned using standard procedures. Mutations can then be introduced into the Npro protease gene by procedures such as the polymerase chain reaction (PCR), site directed mutagenesis, by synthesizing and ligating DNA fragments in such a manner that Npro is partially or completely eliminated, or by random mutagenesis techniques including, e.g., exposure to a chemical mutagen or radiation as known in the art, or by a combination of such procedures.
Once the BVD viral genome has been modified so that the Npro gene is inactivated, it can be cloned into an appropriate vector and produced in large amounts. As discussed above, vectors should include the BVD sequence as a distinct element with a sequence comprising, or consisting essentially of, that of the mutated wild type virus. Either the mutated BVD genome or the vector comprising the genome can be transformed or transfected into a host cell for the purpose of making either large amounts of viral nucleic acid or virus itself.
As discussed above in connection with the BVDdN1 genomic DNA, antibody to BVD virus can be produced in an animal by administering any wild type BVD viral genome that has been mutated in the manner discussed above. In general, it is preferred that antibody production take place in cattle, but other animals can be used as well.
Vaccines incorporating the mutated BVD genomic nucleic acid can be produced and used to induce an immune response in cattle using standard DNA immunization procedures (e.g., those discussed in U.S. Pat. Nos. 5,589,466; 5,580,859; and 5,703,055). The vaccines, antibodies, and nucleic acids made by the methods discussed herein are all part of the present invention.
D. Methods of Making Attenuated BVD Virus
It has been discovered that when the nucleic acid of a BVD virus is mutated so as to inactivate the Npro protease gene, an attenuated virus is produced that is much less infectious in cell culture. The relatively slow replication of these attenuated viruses allows animals to marshal their immunological defenses in a way that is not possible for a rapidly propagating wild type virus. Thus, the methods for producing a mutated viral genome discussed above for BVDdN1 lead directly to a general method for attenuating BVD virus so as to make it suitable for use in a vaccine. In general, the procedure involves isolating a wild type BVD virus; cloning its genomic nucleic acid; mutating the cloned nucleic acid so as to inactivate the Npro protease gene; and then transforming or transfecting the mutated nucleic acid into a host to produce the attenuated virus. Although any of the methods discussed above for producing mutations can be used, the preferred method will be to delete all or part of the Npro protease gene.
The present invention encompasses not only methods for making attenuated virus, but also the virus itself, host cells infected with the virus and progeny virus produced by these host cells. Antibody can be made to the attenuated BVD virus by infecting animals, preferably cattle, at an effective dosage. Antibodies made in this manner are part of the invention and can be isolated and used in diagnostic procedures, or for detecting the presence of BVD in cell culture.
As discussed in connection with the BVDdN1 virus, attenuated virus characterized by an inactivated Npro protease gene can be incorporated into vaccines and used to induce an immune response in cattle. Dosages and immunization protocols can be optimized so that inoculation of animals results in protective immunity against subsequent viral challenge.